Salmonella sp are facultative intracellular bacteria that reside and replicate within a vacuole (SCV). The biogenesis of the SCV is determined by the bacteria and involves effector proteins that are translocated into the host cell via Type III secretion systems. Invasion occurs as a result of localized membrane ruffles that are due to local rearrangement of the actin cytoskeleton. This process is induced by the co-operative effectives of several translocated effector proteins. Membrane ruffles contain large amounts of membrane that must be rapidly delivered. It has been assumed, since only endocytic markers are detected in SCVs, that the biosynthetic/secretory pathway is not involved in this process. In collaboration with Michel Desjardins at the University of Montreal, we have shown that the SCV rapidly acquires Endoplasmic Reticulum membranes and indeed appears contiguous with the ER. Thus it is possible that the ER is contributing membrane content to ruffles at the plasma membrane. Indeed, in phagocytic cells, ER membrane can be seen to be contiguous with nascent phagosomes containing either latex beads or Leishmania. We have developed a live cell system that we are using to characterize 3 stages of Salmonella-host cell interactions; invasion, SCV formation and Sif dynamics. Sifs are tubules that extend from, and are contiguous with, SCVs. The formation of Sifs is dependent on several Salmonella effector proteins and unidentified host factors. We have found that these extensive tubules are extremely dynamic and are dependent on microtubules. Further characterization of the role of microtubules, motors and other host cell factors is ongoing. In particular we are using small inhibitory RNAs to deplete host cell proteins that may play a role in SCV/sif formation.